Development in metazoans is regulated by specific regulatory hierarchies leading to the stage- and tissue-specific expression of transcriptional networks. Understanding such networks poses a major challenge in developmental biology. A model is provided by the regulation of sexual dimorphism in Drosophila melanogaster. This collaborative R01 application from the laboratories of M. Weiss (CWRU) and B. Baker (Stanford) focuses on Doublesex (DSX), a member of a newly recognized family of DM transcription factors broadly involved in metazoan development, including in humans. Our long-term goal is a molecular understanding of how information is transmitted from the primary determinants of sex to the genes responsible for the morphological, biochemical, and behavioral differences between the sexes. The doublesex (dsx) gene controls a major branch of the sex-determining hierarchy of D. melanogaster. The doublesex (dsx) gene encodes sex-specific protein isoforms (designated DSXF (female) and DSXM (male)) as a consequence of sex-specific RNA splicing. Although DSXF and DSXM have opposing biological functions, they exhibit similar DNA-binding properties and so presumably recognize the same cis- acting control elements in target genes. DSXF (but not DSXM) interacts with transcriptional coactivator Intersex (IX), presumably leading to assembly of a female-specific multiprotein-DNA complex at target genes. Such sex-specific regulatory properties presumably underlie the function of dsx as a prototypical "behavioral gene" in the nervous system: DSXM regulates sine singing, a component of the male courtship song, whereas DSXF is hypothesized to regulate major features of female courtship behavior, including rejection behavior. We seek to investigate the molecular bases of the sex-specific functions of DSXF and DSXM with application to behavioral genetics. To these ends, an innovative interdisciplinary strategy is proposed that integrates molecular genetics with biochemistry, proteomics, and structural biology. Studies will focus on the respective C-terminal domains of DSXF/M containing sex-specific tails. In addition, newly described methods for gene targeting in vivo will be employed to investigate the relationship between dsx as a behavioral gene and a discrete and quantifiable repertoire of behavioral elements. Because deletions of DSX-related genes in human chromosome 9 are associated with intersexual abnormalities of the newborn, our results may also provide insight into a major class of human birth defects. Public Health Relevance: How the differences between male and female animals are specified by genes defines a major scientific problem. We are studying sex-specific genes in fruit flies to gain insight into human birth defects, infertility syndromes, and cancers of the gonads.